RESUMO
The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROOâ¢) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROOâ¢, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROOâ¢. Enzyme mixtures (1:1:1 ratio) were exposed to ROO⢠produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated â¼175 µM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (â¼100 µM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.
Assuntos
Escherichia coli , Glucosefosfato Desidrogenase , NADP , Oxirredução , Via de Pentose Fosfato , Fosfogluconato Desidrogenase , Glucosefosfato Desidrogenase/metabolismo , Fosfogluconato Desidrogenase/metabolismo , NADP/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Ribulosefosfatos/metabolismo , Glucose-6-Fosfato/metabolismo , Peróxidos/metabolismo , Hidrolases de Éster CarboxílicoRESUMO
Giardia lamblia, due to the habitat in which it develops, requires a continuous supply of intermediate compounds that allow it to survive in the host. The pentose phosphate pathway (PPP) provides essential molecules such as NADPH and ribulose-5-phosphate during the oxidative phase of the pathway. One of the key enzymes during this stage is 6-phosphogluconate dehydrogenase (6 PGDH) for generating NADPH. Given the relevance of the enzyme, in the present work, the 6pgdh gene from G. lamblia was amplified and cloned to produce the recombinant protein (Gl-6 PGDH) and characterize it functionally and structurally after the purification of Gl-6 PGDH by affinity chromatography. The results of the characterization showed that the protein has a molecular mass of 54 kDa, with an optimal pH of 7.0 and a temperature of 36-42 °C. The kinetic parameters of Gl-6 PGDH were Km = 49.2 and 139.9 µM (for NADP+ and 6-PG, respectively), Vmax =26.27 µmol*min-1*mg-1, and Kcat = 24.0 s-1. Finally, computational modeling studies were performed to obtain a structural visualization of the Gl-6 PGDH protein. The generation of the model and the characterization assays will allow us to expand our knowledge for future studies of the function of the protein in the metabolism of the parasite.
Assuntos
Giardia lamblia/enzimologia , Gluconatos/química , NADP/química , Fosfogluconato Desidrogenase/química , Proteínas de Protozoários/química , Ribulosefosfatos/química , Motivos de Aminoácidos , Sítios de Ligação , Clonagem Molecular/métodos , Expressão Gênica , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/enzimologia , Giardia lamblia/genética , Gluconatos/metabolismo , Humanos , Cinética , Modelos Moleculares , NADP/metabolismo , Via de Pentose Fosfato/genética , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulosefosfatos/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , TermodinâmicaRESUMO
The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.
Assuntos
Proteínas de Bactérias/metabolismo , Gluconacetobacter/enzimologia , Gluconatos/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Ribulosefosfatos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Gluconacetobacter/genética , Gluconatos/química , Humanos , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , NAD/metabolismo , NADP/metabolismo , Fosfogluconato Desidrogenase/classificação , Fosfogluconato Desidrogenase/genética , Filogenia , Domínios Proteicos , Multimerização Proteica , Ribulosefosfatos/química , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Efficient xylose fermentation still demands knowledge regarding xylose catabolism. In this study, metabolic flux analysis (MFA) and metabolomics were used to improve our understanding of xylose metabolism. Thus, a stoichiometric model was constructed to simulate the intracellular carbon flux and used to validate the metabolome data collected within xylose catabolic pathways of non-Saccharomyces xylose utilizing yeasts. RESULTS: A metabolic flux model was constructed using xylose fermentation data from yeasts Scheffersomyces stipitis, Spathaspora arborariae, and Spathaspora passalidarum. In total, 39 intracellular metabolic reactions rates were utilized validating the measurements of 11 intracellular metabolites, acquired by mass spectrometry. Among them, 80% of total metabolites were confirmed with a correlation above 90% when compared to the stoichiometric model. Among the intracellular metabolites, fructose-6-phosphate, glucose-6-phosphate, ribulose-5-phosphate, and malate are validated in the three studied yeasts. However, the metabolites phosphoenolpyruvate and pyruvate could not be confirmed in any yeast. Finally, the three yeasts had the metabolic fluxes from xylose to ethanol compared. Xylose catabolism occurs at twice-higher flux rates in S. stipitis than S. passalidarum and S. arborariae. Besides, S. passalidarum present 1.5 times high flux rate in the xylose reductase reaction NADH-dependent than other two yeasts. CONCLUSIONS: This study demonstrated a novel strategy for metabolome data validation and brought insights about naturally xylose-fermenting yeasts. S. stipitis and S. passalidarum showed respectively three and twice higher flux rates of XR with NADH cofactor, reducing the xylitol production when compared to S. arborariae. Besides then, the higher flux rates directed to pentose phosphate pathway (PPP) and glycolysis pathways resulted in better ethanol production in S. stipitis and S. passalidarum when compared to S. arborariae.
Assuntos
Fermentação , Análise do Fluxo Metabólico/métodos , Metaboloma , Metabolômica/métodos , Saccharomycetales/metabolismo , Frutosefosfatos/metabolismo , Glucose-6-Fosfato/metabolismo , Glicólise , Malatos/metabolismo , Espectrometria de Massas/métodos , Modelos Biológicos , Via de Pentose Fosfato , Ribulosefosfatos/metabolismo , Saccharomycetales/classificação , Leveduras/classificação , Leveduras/metabolismoRESUMO
The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/ß TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these structural findings.
Assuntos
Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Ribulosefosfatos/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Carboidratos Epimerases/genética , Catálise , Clonagem Molecular , Isoenzimas , Cinética , Modelos Moleculares , Conformação Proteica , Homologia de Sequência de Aminoácidos , Frações SubcelularesRESUMO
Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for MtIGPS-catalized chemical reaction is proposed.
Assuntos
Indol-3-Glicerolfosfato Sintase/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Bases , Fenômenos Biofísicos , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Indol-3-Glicerolfosfato Sintase/genética , Indol-3-Glicerolfosfato Sintase/isolamento & purificação , Cinética , Espectroscopia de Ressonância Magnética , Modelos Químicos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Ribulosefosfatos/síntese química , Ribulosefosfatos/metabolismo , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Termodinâmica , VirulênciaRESUMO
Photosynthetic responses of sunflower plants grown for 52 d in ambient and elevated CO(2) (A=350 or E=700 micromol mol(-1), respectively) and subjected to no (control), mild or severe water deficits after 45 d were analysed to determine if E modifies responses to water deficiency. Relative water content, leaf water potential (Psi(w)) and osmotic potential decreased with water deficiency, but there were no effects of E. Growth in E decreased stomatal conductance (g(s)) and thereby transpiration, but increased net CO(2) assimilation rate (P(n), short-term measurements); therefore, water-use efficiency increased by 230% (control plants) and 380% (severe stress). Growth in E did not affect the response of P(n) to intercellular CO(2) concentration, despite a reduction of 25% in Rubisco content, because this was compensated by a 32% increase in Rubisco activity. Analysis of chlorophyll a fluorescence showed that changes in energy metabolism associated with E were small, despite the decreased Rubisco content. Water deficits decreased g(s) and P(n): metabolic limitation was greater than stomatal at mild and severe deficit and was not overcome by elevated CO(2). The decrease in P(n) with water deficiency was related to lower Rubisco activity rather than to ATP and RuBP contents. Thus, there were no important interactions between CO(2) during growth and water deficit with respect to photosynthetic metabolism. Elevated CO(2 )will benefit sunflower growing under water deficit by marginally increasing P(n), and by slowing transpiration, which will decrease the rate and severity of water deficits, with limited effects on metabolism.